The present invention relates to a microscope for conventional fluorescence microscopy (epi-fluorescence) and for total internal reflection microscopy, comprising at least one light source for the conventional fluorescent illumination and at least one light source for the evanescent illumination, and comprising one objective, wherein the illumination light coming from the light sources on different illumination paths is passed via a beam combiner into the objective and from there to the specimen, the beam combiner being structured such that it directs the illumination light used for the conventional fluorescent illumination and the illumination light used for the evanescent illumination into the objective in geometrically separated beam paths.
The total internal reflection microscopy makes use of the refractive behavior of light during the transition from an optically denser medium to an optically thinner medium. Thus, for instance, the transition from cover glass (n1=1.518) to water (n2=1.33) yields a critical angle of 61°, the angle of total reflection. Under the conditions of total reflection (angle ≧61°), a standing evanescent wave forms in the medium having the lower refractive index. The intensity of this wave drops exponentially relative to the distance from the interface. For this reason, fluorophores located far away from the interface are not excited. The background fluorescence is drastically reduced. The image contrast is improved in this process while, at the same time, the resolution is considerably increased. A prerequisite for utilizing the afore-described phenomenon is a sufficiently large difference between the refractive indices of the cover glass and of the medium.
U.S. Pat. Appln. No. US 2002/0097489 A1 discloses a microscope with evanescent illumination of a specimen. The microscope comprises a white-light source whose light is coupled into the specimen slide via a slit aperture through the microscope objective for purposes of evanescent illumination. The illumination light propagates in the specimen slide due to total internal reflection, wherein the specimen is only illuminated in the region of the evanescent field that extends from the specimen slide. Microscopes of this type are known by the acronym TIRFM (Total Internal Reflection Fluorescent Microscope). The z-resolution of TIRF microscopes is exceptionally good owing to the fact that the evanescent field extends only about 100 nm into the specimen.
German patent application DE 101 08 796 A1 discloses a high-aperture objective, particularly for TIRF applications. The objective consists of a first lens having a positive refractive power, a second lens having a negative refractive power, the focal length ratio between the two lenses lying within the range from −0.4 to −0.1, and, the total refractive power being greater than zero. Further, the objective comprises two positive lenses whose diameter-to-focal length ratio is greater than 0.3 and smaller than 0.6. Furthermore, the objective comprises a negative lens and a collective lens, the negative lens facing the front group and the focal length ratio between the negative lens and the collective lens lying between −0.5 and −2.
German patent application DE 102 17 098 A1 discloses an incident-illumination arrangement for TIRF microscopy. The incident-illumination arrangement contains a source of illumination that, during operation, emits a polarized illuminating beam bundle that propagates at an angle relative to the optical axis, as well as a deflecting device that deflects the illuminating beam bundle and couples it into the objective parallel to the optical axis. With this incident-illumination arrangement, it is provided that the illuminating beam bundle emitted by the source of illumination has s-polarization and p-polarization directions with a phase difference and the deflecting device reflects the illuminating beam bundle x times, wherein x=(n×180°−d)/60°.
German patent application DE 101 43 481 A1 discloses a microscope for TIRM (Total Internal Reflection Microscopy). The microscope has a housing and an objective. The illumination light emitted by an illumination device can be coupled in by means of an adapter that can be slid into the microscope housing.
U.S. Pat. Appln. No. US 2004/0001253 A1 discloses a microscope with an optical illumination system that allows a simple switching over between evanescent illumination and reflection illumination. The illumination system comprises a source of laser light whose light is coupled into an optical fiber. Moreover, a coupling-out optical system is provided that focuses the light coming out of the fiber into a rear focal point of the microscope objective. The optical fiber can be moved in a plane perpendicular to the optical axis of the microscope objective.
German patent application DE 102 29 935 A1 discloses a device for the coupling in of light in a microscope. There, laser light is directed onto the preparation in the light field aperture plane by way of an in-coupling light-conducting fiber configured as a slider. The invention is particularly well-suited for the TIRF method.
In scanning microscopy, a specimen is illuminated with a light beam so that the detection light emitted by the specimen can be observed as reflection light or fluorescent light. The focus of an illumination beam bundle is moved in a specimen plane by means of a controllable beam deflector, usually by tilting two mirrors, wherein the deflection axes are usually perpendicular to each other, so that one mirror deflects in the x-direction while the other deflects in the y-direction. The mirrors are tilted by means of, for example, galvanometric actuators. The power of the detection light coming from the object is measured as a function of the position of the scanning beam. Normally, the actuators are fitted with sensors in order to determine the current position of the mirror. Especially in the case of confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light from the light source is focused onto a pinhole (the so-called excitation aperture), a beam splitter, a beam deflector for beam control, a microscope optical system, a detection aperture and the detectors to detect the detection light or fluorescent light. The illumination light is coupled in by means of a beam splitter. The fluorescent light or reflection light coming from the object returns to the beam splitter via the beam deflector, passes through the beam splitter in order to be subsequently focused onto the detection aperture downstream of which the detectors are located. This detector arrangement is called a descan arrangement. Detection light that does not stem directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained that yields a three-dimensional image as a result of sequential scanning of the object with the focus of the illuminating beam bundle. For the most part, a three-dimensional image is obtained by means of layerwise image data acquisition.
With the microscopes known from the prior art, the evanescent illumination is regularly coupled in within the scope of two-dimensional solutions, even if the adjustment unit used in such cases is always configured one-dimensionally. Thus, the coupling-in is done, for instance, by means of a so-called neutral splitter, i.e. by means of a mirror that reflects light to a certain extent and otherwise transmits light. Coupling-in by means of a dichroitic splitter is also known. In this case, it is a special mirror that, except for one specific wavelength, reflects all other wavelengths. Another known approach is the coupling-in by means of a polarization splitter. Here, the lasers for the evanescent illumination (TIRF illumination) and the laser for the conventional epi-fluorescent illumination are polarized orthogonally with respect to each other and then combined. As a one-dimensional possibility for coupling in the requisite source of radiation, it is likewise already known to use small additional mirrors in the illumination beam path for the epi-fluorescent illumination.
The methods and devices known so far for coupling in one or more radiation sources for evanescent illumination are problematic in practice since restrictions on the specific properties of the respectively used radiation source arise from the type of coupling-in. Coupling-in via a neutral splitter has the disadvantage that deteriorations in the performance occur both in the case of the radiation source for the evanescent illumination and also in the case of the radiation source for the epi-fluorescent illumination. Coupling-in via a dichroitic splitter has the disadvantage that a specific wavelength or a specific wavelength range must be specified. If one wishes to change the wavelength within the scope of such a realization, then the beam combiner or mirror has to be changed as well. Coupling-in by means of a polarization splitter also brings with it the serious disadvantage that all components used must be designed as polarization-preserving. Moreover, the use of a polarization splitter means dispensing with a further degree of freedom at the radiation sources. Finally, coupling-in by means of small additional mirrors in the illumination beam path of the epi-fluorescent illumination is out of question right from the beginning because this involves a one-dimensional solution. The additional mirrors moreover result in a partial covering of the epi-fluorescent illumination so that in this regard, too, this possibility for coupling-in is not acceptable.
German patent DE 103 09 269 B4 discloses a device for total internal reflection microscopy, in which for beam combination of the illumination light used for the epi-fluorescent illumination as well as the illumination light used for the evanescent illumination a beam combiner is provided which is structured such that it has an outer transmissive area for the illumination light used for the evanescent illumination as well as an inner reflective area for the illumination light used for the conventional fluorescent illumination. What is disadvantageous is that the areas of the beam combiner have to be precisely adapted to the objective used so that a change of the objective without a simultaneous change of the beam combiner is not possible. In this respect, each change of objective requires enormous adjustment work.